Temozolomide is an alkylating agent that has displayed pre-clinical activity against a broad spectrum of murine tumours in vivo (Stevens M G F, 1987). In vitro studies have demonstrated activity against a wide variety of tumours including some usually resistant to chemotherapy with more established drugs (Raymond E, 1997). It is currently indicated for the treatment of malignant glioma and is available as a capsule for oral administration in a number of countries including US and Europe. Phase II trials of temozolomide have confirmed that it has significant activity in patients with metastatic malignant melanoma (Bleehen N M, 1995). More recently, a phase III trial indicated that temozolomide is as effective as dacarbazine in patients with advanced metastatic melanoma with progression-free survival favouring temozolomide (Middleton M R, 2000). Prolonged exposure, using an extended dosing schedule, has been suggested to increase clinical activity but has led to an unusually high level of side-effects. Major side-effects of temozolomide include leukopenia, nausea, vomiting with alopecia, rash and constipation to a lesser degree. With extended dosing, there was also an increased incidence of opportunistic infections. In addition, temozolomide has been shown to have dose-limiting myelotoxicity following oral delivery. Therefore, it is very important to develop other potential routes of delivery to maximize temozolomide therapeutical potential. However, poor solubility in both aqueous and organic media and instability of the solubilised form, have resulted in serious difficulties for pharmaceutical studies and exploration of alternative delivery routes. As a result, only a limited number of studies have been carried out with attempts to reduce the toxicity via intrathecal delivery of a solubilised formulation (Sampson J. H, 1999) and intracerebral microinfusion of temozolomide (Heimberger A. M, 2000)
The development of new therapeutic agents and new drug delivery strategies is of very important for treating cancers. Skin cancers (melanomas) are one of most difficult to treat cancers because they are very aggressive and resistant to general chemotherapy. In order to maximise temozolomide efficiency and to improve its therapeutic benefit for curing skin cancer, particularly in the early stages, transdermal delivery may fulfill the object. It has been showed that topically applied toremifene, an oestrogen-receptor antagonist licensed for treatment of breast cancer, achieved high local concentrations of drug in the tumours, with low systemic levels when investigated as a melanoma treatment (Maenpaa, J, 1993 and Soe L, 1997). Dermal delivery of drugs is greatly limited by the efficient skin barrier and the generally unfavourable physicochemical properties of drugs. Synthesis of an ester prodrug is one method to enhance the skin absorption of a topically applied drug where, by chemical modification of the penetrating molecule, its delivery can be enhanced. The epidermal layer contains many non-specific enzymes that are capable of hydrolysing carboxylate bonds to release the parent compound (Valia K. H. 1985). An example of such an approach is the esterification of poorly skin-permeable estradiol to form lipophilic estradiol esters. The study showed that all the estradiol esters investigated were extensively metabolised in the skin during permeation to regenerate the biologically active parent. Some esters were shown to achieve permeation rates two-to-four fold higher than free estradiol. A series of 1,3-bisalkylcarbonyl-5-flurouracil prodrugs have been investigated for topical delivery (Beall H D, 2002).
Temozolomide spontaneously degrades in physiological fluid to generate the cytotoxic methylating agent, 5(3-methyl-1-triazeno) imidazole-4-corboxamide, which subsequently fragments to the DNA-methylating agent, methyldiazonium. Tsang (Tsang, L L H 1990) in his study of urinary metabolites of temozolomide described in vitro IC50 of 8-(carboxylic acid)-3-methylimidazo[5,1-d]-1,2,3,5-tetrazine-4(3H)-one (temozolomide acid) was almost identical to that of temozolomide against TLX5 lymphoma cell line. In light of this report, we decided to conduct experiments to verify whether or not the temozolomide acid is as active as temozolomide against melanoma and glioma. In in vitro bioactivity tests, the temozolomide acid was used against a selected panel of cancer cell lines including melanoma and glioma. The experiments demonstrated the temozolomide acid is cytotoxic against certain melanoma and glioma cells and the IC50s of the temozolomide acid and temozolomide are in the same grade. Therefore, the temozolomide acid is the parental active drug with the same indications to temozolomide and the temozolomide ester prod-rug approach is the feasible strategy for maximizing temozolomide therapeutic benefit for the treatments of cancers, particularly for skin cancer through topical or transdeermal application.
The past studies of structural optimization of temozolomide mainly concentrated on modification of an alkyl group on N3 and an alkyl group on N of 8-carbamoyl. Though the general formula of EP 0252682 covered temozolomide methyl, ethyl, propyl and butyl esters, no synthetic methods and physical and chemistry data for these esters were included, except a benzyl ester. U.S. Pat. No. 5,260,291, GB 2104522, GB 2125402, FR 2511679 and FR 2531958 only covered temozolomide and its 8-carboxylamide derivatives. Temozolomide ethyl ester was reported as a precursor for studies of new synthetic routes for temozolomide (Wang Y, 1997).
In conclusion, neither a research aiming to develop a topical or a transdermal administration of temozolomide and its derivatives through design and synthesis of a series of temozolomide esters nor a general synthesis for temozolomide esters have been reported. Furthermore, there is no report about enzymatically metabolizing temozolomide esters into the bioactive temozolomide acid in skins and potential application of the temozolomide esters as pro-drugs in treating cancers.